This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. NANOG, Sox2 and Oct4 are transcription factors with key functions in mammalian stem cell biology. They are central of pluripotency in ES cells. Loss of NANOG induces differentiation towards an endoderm-like lineage in embryonic stem (ES) cells, whereas forced expression retains the ES phenotype in the absence of the otherwise essential growth factors. NANOG binds to promoter elements of hundreds of target genes and regulates their expression. The mechanism of this transcriptional regulation is not yet known. Currently, one X-ray crystal structure of the homeodomain of murine Nanog is available in the PDB;however, numerous studies have shown that the pathways maintaining pluripotency are different between human and mouse ES cells. We plan to determine the structure and DNA binding studies of NANOG, Sox2 and Oct4 by NMR.